The ultimate objective of this research program is to understand how androgens modulate brain function and regulate the expression of male sexual behavior (MSB). Our approach is to employ biochemical and molecular techniques in combination with behavioral studies to address questions on androgen action in male rat brain. We have expanded our behavioral investigations to include androgen-dependent measures such as partner preference, vocalization and anogenital investigation. All of these behaviors, along with copulation itself, are influential in the expression of MSB and will be referred to herein as androgen-dependent sexual behaviors. To achieve our goals, we will investigate three questions. Question l focusses on the neural sites of androgen action in regulating androgen-dependent sexual behaviors. The androgen receptor (AR) blocker, hydroxyflutamide (OHF), will be used to assess the role of AR in mediating androgen-dependent sexual behaviors in two previously unstudied androgen- concentrating brain sites: the ventromedial hypothalamic nucleus (VMN) and cranial nerve motor nuclei (n. ambiguous, hypoglossal n., facial motor n.). Question 2 focusses on the controversy over whether MSB is mediated by testosterone (T) acting via AR, or by aromatization to estradiol (E2). We will extend our recent results showing that both T and E2 are essential for MSB, by employing a newER blocker, RU58668, to assess the role of estrogen receptors (ER) in mediating androgen-dependent sexual behaviors. The site specificity of E2 action will be determined by implanting RU58668 directly into the medial preoptic area (MPOA), VMN or medial amygdala. Question 3 focusses on the identification of androgen-dependent genes in the hypothalamus-preoptic area (HYP-POA). We will use a subtractively hybridized probe to screen our HYP-POA cDNA library from T-treated adult male rats. RNAse protection assays will be used to verify that the isolated clones are androgen-dependent. Confirmed androgen- dependent clones will be sequenced to establish their identity. The answers to the three questions posed above are central to determining the consequences of T exposure in critical brain sites necessary for MSB, to resolving the controversy over the specificity of T action in the expression of androgen-dependent sexual behaviors, and will provide new insights into the cellular effects of T exposure. The results will also provide important knowledge on the effects of AR and ER blockers on behavior, which has clinical relevance for treating patients suffering from hormone-dependent tumors or from hormonal or sexual dysfunction.